The invention relates to cyanine dyes for use as fluorescent markers.
Fluorescent dyes are used as markers in many different ways for the analysis of a major part of clinically, biologically, biochemically or chemically relevant substances, such as for example cells, antibodies, proteins, hormones, nucleic acids, oligonucleotides, carbohydrates or amines and mercaptanes. In combination with the easily detectable fluorescence induced by laser (LIF; Laser-Induced Fluorescence), rapid analyses are possible in the aforementioned areas.
By means of covalent bonding, a larger number of dye molecules are able to be attached to a specimen than is possible in the case of a non-specific non-covalent bond, so that the intensity of the fluorescent light signal is stronger. Furthermore, a covalent bond enables unequivocal marking of a specific target structure in a mixture. For covalent bonding of the fluorescent dye to the specimen, therefore, the dyes are provided with reactive groups. Suitable reactive groups are, for example, the iodacetamid group, the isothiocyanate group, N-succinimide-esters of alkyl carboxylic acids or phosophoramidites of hydroxy alkyl groups.
In the case of antibodies, proteins, hormones, nucleic acids and other biomolecules, mercapto (xe2x80x94SH), amino (xe2x80x94NH2) and hydroxy groups (xe2x80x94OH), among others, are available.
Cyanine dyes are increasingly being used as fluorescent markers. Generally, however, cyanine dyes in solution and at surfaces tend to form aggregates [SCHEIBE: Angew. Chem. 49 (1936) 563; JELLEY: Nature 138 (1936) 1009]. These polymeric dye systems, in contrast with the individual molecule, exhibit a markedly changed absorption and fluorescence behaviour.
As a result of the aggregation, the absorption maximum may be shifted in comparison with the monomer hypsochrome (Dimers, H-aggregates) or bathochrome (J-aggregates) [DIETZE: J. Signal AM. 1 (1973) 157]. This different absorption behaviour depends on the different dislocation angle between the molecule and aggregate axis as well as the intermolecular spacing of the dyes molecules in the aggregate (packing density). Independently of these influencing variables, it was found that at least seven dye molecules are needed for the formation of J-aggregates [DALTROZZO et al.: Photogr. Sci. Eng. 20 (1974) 441], i.e. J-aggregates are only able to form with raised packing density.
It is important when using cyanine dyes as fluorescent markers in aqueous systems to prevent the formation of dye aggregates, which fluoresce much more poorly. In addition, there is the fact that the tendency towards increased dye aggregation is accompanied by an increased tendency towards non-specific interactions of the dye at an arbitrary biomolecule.
Aryl sulpho groups lead to a marked improvement in the water solubility of a dye and should at the same time prevent the formation of dimers or H-aggregates. Corresponding fluorescent markers are described in DE 3.912.046, US 5.268.486 and [MUJUMDAR et al.: Bioconjugate Chem. 4 (1993) 105]. 
Using the example of the dye with the following structure 
it could be shown that J-aggregates form in aqueous solution, whereas coupled with proteins, a very marked dimer band could be detected [MUJUMDAR et al.: Bioconjugate Chem. 7 (1996) 356], as a result of which a considerable fluorescence quenching occurs. It goes on to describe how the dimer formation is suppressed by the introduction of two further sulpho groups. 
It has been shown, however, that the fluorescence quantum yield of the protein conjugates both of dye 1 and also 3 decreases by around xc2xc compared with free, unconjugated dyes [MUJUMDAR et al.: Bioconjugate Chem. 7 (1996) 356; Terpetschnig: NATO Advanced Research Workshop, Conference Paper, Trieste, 1997].
The object of the invention is to provide new cyanine dyes which do not exhibit the aforementioned drawbacks and which in particular exhibit a low tendency towards aggregation both in solution as well as on the target molecule and are suitable for application as fluorescent markers in the range from 500 to 700 nm.
This object is solved by a cyanine dye with the features according to claim 1. Advantageous embodiments and applications emerge from the dependent claims.